Pressing Too Hard on Transfer Sandwich - (Mar/03/2016 )
I've been struggling to get clear western blot bands despite following standard protocols to the tee.
One comment that has come up is I press down hard on my western blot sandwich before transferring to get rid of the air bubbles. Could protein transfer just by force alone? Has anyone had experience with this cause smearing on western blots?
Proteins start transferring as soon (using capillary effect) as soon as you place the membrane on the gel. Pressing too hard could squash the gel and cause it to re-position, so that the final transfer is smeared. If you lay the membrane down from one edge, it should be possible to place a curve in the membrane that will help remove bubbles, then a light roll will be sufficient to remove any others.
Also, make sure the membrane is wet, which will slow down capillary transfer.
Try to use more buffer during you making the sandwich, and you could see whether there is bubbles between gel and PVDF.